Lorenzo Bermejo, Teresa

To investigate the influence of geographic constrains to mobility on SARS-CoV-2 circulation before the advent of vaccination, we recently characterized the occurrence in Sicily of viral lineages in the second pandemic wave (September to December 2020). Our data revealed wide prevalence of the then widespread through Europe B.1.177 variant, although some viral samples could not be classified with the limited Sanger sequencing tools used. A particularly interesting sample could not be fitted to a major variant then circulating in Europe and has been subjected here to full genome sequencing in an attempt to clarify its origin, lineage and relations with the seven full genome sequences deposited for that period in Sicily, hoping to provide clues on viral evolution. The obtained genome is unique (not present in databases). It hosts 20 single-base substitutions relative to the original Wuhan-Hu-1 sequence, 8 of them synonymous and the other 12 encoding 11 amino acid substitutions, all of them already reported one by one. They include four highly prevalent substitutions, NSP12:P323L, S:D614G, and N:R203K/G204R; the much less prevalent S:G181V, ORF3a:G49V and N:R209I changes; and the very rare mutations NSP3:L761I, NSP6:S106F, NSP8:S41F and NSP14:Y447H. GISAID labeled this genome as B.1.1 lineage, a lineage that appeared early on in the pandemic. Phylogenetic analysis also confirmed this lineage diagnosis. Comparison with the seven genome sequences deposited in late 2020 from Sicily revealed branching leading to B.1.177 in one branch and to Alpha in the other branch, and suggested a local origin for the S:G118V mutation.

In the setting of the recent COVID-19 pandemic, transmission of SARS-CoV-2 to animals has been reported in both domestic and wild animals and is a matter of concern. Given the genetic and functional similarities to humans, non-human primates merit particular attention. In the case of lemurs, generally considered endangered, they are believed to be susceptible to SARS-CoV-2 infection. We have conducted a study for evidence of SARS-CoV-2 infection among the 43 lemurs of Mundomar, a zoological park in Benidorm, Spain. They belong to two endangered lemur species, 23 black-and-white ruffed lemurs (Varecia variegata) and 20 ring-tailed lemurs (Lemur catta). Health assessments conducted in 2022 and 2023 included molecular analyses for SARS-CoV-2 RNA of oral and rectal swabs using two different RT-qPCR assays, always with negative results for SARS-CoV-2 in all animals. The assessment also included serological testing for antibodies against the receptor-binding domain (RBD) of the spike protein (S) of SARS-CoV-2, which again yielded negative results in all animals except one black-and-white ruffed lemur, supporting prior infection of that animal with SARS-CoV-2. Our data, while not indicating a high susceptibility of lemurs to SARS-CoV-2 infection, show that they can be infected, adding to the existing information body on potential ways for SARS-CoV-2 virus spreading in zoos, highlighting the need for animal surveillance for the virus.

To provide new insight into the pathophysiological mechanisms underlying gas emboli (GE) in bycaught loggerhead sea turtles (Caretta caretta), we investigated the vasoactive characteristics of the pulmonary and systemic arteries, and the lung parenchyma (LP). Tissues were opportunistically excised from recently dead animals for in vitro studies of vasoactive responses to four different neurotransmitters: acetylcholine (ACh; parasympathetic), serotonin (5HT), adrenaline (Adr; sympathetic) and histamine. The significant amount of smooth muscle in the LP contracted in response to ACh, Adr and histamine. The intrapulmonary and systemic arteries contracted under both parasympathetic and sympathetic stimulation and when exposed to 5HT. However, proximal extrapulmonary arterial (PEPA) sections contracted in response to ACh and 5HT, whereas Adr caused relaxation. In sea turtles, the relaxation in the pulmonary artery was particularly pronounced at the level of the pulmonary artery sphincter (PASp), where the vessel wall was highly muscular. For comparison, we also studied tissue response in freshwater sliders turtles (Trachemys scripta elegans). Both PEPA and LP from freshwater sliders contracted in response to 5HT, ACh and also Adr. We propose that in sea turtles, the dive response (parasympathetic tone) constricts the PEPA, LP and PASp, causing a pulmonary shunt and limiting gas uptake at depth, which reduces the risk of GE during long and deep dives. Elevated sympathetic tone caused by forced submersion during entanglement with fishing gear increases the pulmonary blood flow causing an increase in N2 uptake, potentially leading to the formation of blood and tissue GE at the surface. These findings provide potential physiological and anatomical explanations on how these animals have evolved a cardiac shunt pattern that regulates gas exchange during deep and prolonged diving.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has infected several animal species, including dogs, presumably via human-to-animal transmission. Most infected dogs reported were asymptomatic, with low viral loads. However, in this case we detected SARS-CoV-2 in a dog from the North African coastal Spanish city of Ceuta presenting hemorrhagic diarrhea, a disease also reported earlier on in an infected dog from the USA. Case presentation: In early January 2021, a West Highland Terrier pet dog from Ceuta (Spain) presented hemorrhagic diarrhea with negative tests for candidate microbial pathogens. Since the animal was in a household whose members suffered SARS-CoV-2 in December 2020, dog feces were analyzed for SARS-CoV-2, proving positive in a twotube RT-PCR test, with confirmation by sequencing a 399-nucleotide region of the spike (S) gene. Furthermore, nextgeneration sequencing (NGS) covered > 90% SARS-CoV-2 genome sequence, allowing to classify it as variant B.1.177. Remarkably, the sequence revealed the Ile402Val substitution in the spike protein (S), of potential concern because it mapped in the receptor binding domain (RBD) that mediates virus interaction with the cell. NGS reads mapping to bacterial genomes showed that the dog fecal microbiome fitted best the characteristic microbiome of dog’s acute hemorrhagic diarrhea. Conclusion: Our findings exemplify dog infection stemming from the human SARS-CoV-2 pandemic, providing nearly complete-genome sequencing of the virus, which is recognized as belonging to the B.1.177 variant, adding knowledge on variant circulation in a geographic region and period for which there was little viral variant characterization. A single amino acid substitution found in the S protein that could have been of concern is excluded to belong to this category given its rarity and intrinsic nature. The dog’s pathology suggests that SARS-CoV-2 could affect the gastrointestinal tract of the dog.